Nalpha, nalpha, nalpha-trialkyl histidine derivatives useful for the preparation of ergothioneine compounds

ABSTRACT

Provided herein are N α ,N α ,N α -trialkyl histidine derivative compounds and methods of their preparation. Also provided are methods of their use for preparing useful compounds such as ergothioneine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application No. 62/133,165,entitled “N^(α), N^(α)-Trialkyl Histidine Derivatives Useful For ThePreparation Of Ergothioneine Compounds,” filed Mar. 13, 2015, and toU.S. Application No. 62/173,895, entitled “N^(α), N^(α), N^(α)-TrialkylHistidine Derivatives Useful For The Preparation Of ErgothioneineCompounds,” filed Jun. 10, 2015; the contents of each application areincorporated by reference herein in their entirety as if put forth fullybelow.

FIELD

Provided herein are compounds and methods useful for the preparation ofcertain betaines such as ergothioneine. Also provided herein are methodsof preparing the compounds and methods of their use for the preparationof certain betaines such as ergothioneine.

BACKGROUND

Ergothioneine was discovered at the beginning of the 20^(th) century inan ergot fungus contaminant of rye. Tenret, 1909, Comp. Rend. Acad. Sci.149:222-224. The compound ergothioneine has been shown to protect cellsfrom reactive oxidative species in bacteria, fungi, plants, and animals,including humans. Paul & Snyder, 2010, Cell Death Differ. 17:1134-1140;Emani et al., 2013, Antimicrob. Agents Chemother. 57:3202-3207. However,only certain bacteria and certain fungi have been shown to produce thecompound themselves. Fahey, 2001, Annu. Rev. Microbiol. 55:333-356.Plants acquire ergothioneine from microbes in the soil. Audley & Tan,1968, Phytochemistry 7:1999-2000. Animals, including humans, absorbergothioneine from their diet. Humans have a specific transporter, ETT,for the uptake of ergothioneine. Gründemann et al., 22005012, Proc.Natl. Acad. Sci. USA 102:5256-5261. Cells that express ETT accumulateand retain ergothioneine at high levels. Id. The compound has a longbiological half-life in the cell. Wolf et al., 1961, Biochem. Biophys.Acta 54:287-293. It is believed that ergothioneine provides antioxidantcytoprotection in such cells. Paul & Snyder, supra.

To date, ergothioneine has been proposed and used in several human andanimal products. These include use as a skin toner additive (U.S. Pat.No. 7,122,211), as a photoprotective agent for human skin (U.S. Pat. No.7,022,317), for the amelioration of liver disease (U.S. Pat. No.6,555,141), for preventing diseases associated with oxidative damage tomitochondria (U.S. Pat. No. 6,479,533; U.S. Pat. No. 6,103,746), forrepair of skin connective tissue damage (U.S. Pat. No. 6,451,771), andfor other pharmaceutical antioxidant uses (U.S. Pat. No. 6,326,034; U.S.Pat. No. 6,056,965).

However, synthesis of ergothioneine has proved difficult and expensive.Commercial sources of ergothioneine can cost tens of thousands ofdollars per gram. Prior syntheses of ergothioneine have involved manysteps, challenging yields, and potentially toxic reagents. See, e.g.,U.S. Pat. Nos. 5,438,151, 7,767,826, and 8,399,500. Improved methods forpreparing ergothioneine on an industrial scale are needed.

SUMMARY

Provided herein are N^(α),N^(α),N^(α)-trialkyl histidine derivativecompounds and methods for their preparation. Also provided herein aremethods of using the N^(α),N^(α),N^(α)-trialkyl histidine derivativecompounds for the preparation of certain betaines such as ergothioneine.Ergothioneine is useful for protecting cells from reactive oxidativespecies in plants and animals, including humans.

In one aspect, provided herein are methods for the preparation of acompound according to Formula 3:

or a salt thereof. In Formula 3, each R¹ can be (C₁₋₄)alkyl. In themethods, the starting material is a compound according to Formula 1:

or a salt or tautomer thereof. In Formula 1, each R¹ can be (C₁₋₄)alkyl.In particular embodiments, the compound of Formula 1 is hercynine (i.e.[(1S)-1-carboxy-2-(1H-imidazol-5-yl)ethyl]-trimethylazanium). In themethods, the compound according to Formula 1 is reacted with achloroformate of formula (benzyl)-OC(O)X where X is halo to open theimidazole ring and yield the compound of Formula 3. Those of skill inthe art will recognize that the methods use the Bamberger imidazolecleavage reaction. Certain methods provided herein are based at least inpart on the discovery that the Bamberger imidazole cleavage reaction canbe applied to a quaternary amine according to Formula 1 withoutresulting in racemization.

In another aspect, provided herein are methods for the preparation of acompound according to Formula 4:

or a salt thereof. In Formula 4, each R¹ can be (C₁₋₄)alkyl. In themethods, the starting material is a compound according to Formula 3,above, or a salt thereof. In one embodiment, the compound according toFormula 3 is transformed by hydrogenolysis using a catalyst, such asPd/C, and hydrogen gas to yield the compound of Formula 4. In anotherembodiment, the compound of Formula 3 is transformed to a compound ofFormula 4 by hydrolysis and rearrangement with an acid, for example, amineral acid such as hydrochloric acid.

In another aspect, provided herein are methods for the preparation of acompound according to Formula 5:

or a salt or tautomer thereof. In Formula 5, each R¹ can be (C₁₋₄)alkyl.In particular embodiments, the compound of Formula 5 is ergothioneine.In the methods, the starting material is a compound according to Formula4, above, or a salt thereof. In the methods, the compound according toFormula 4 is reacted with a thiocyanate to yield the compound of Formula5.

In another aspect, provided herein are compounds according to Formula 3:

and salts thereof. In Formula 3, each R¹ can be (C₁₋₄)alkyl. Compoundsof Formula 3 are useful, for example, for the preparation of certainbetaines such as ergothioneine.

The above methods provide a robust three-step synthesis of compoundssuch as ergothioneine from starting materials such as hercynine which iseasily obtained using methods known in the art (e.g., Reinhold et al.,“Synthesis of α-N-methylated Histidines,” J. Med. Chem. 1968, 11(2), pp.258-260). In addition, the methods provided can be done on an industrialscale. In one embodiment, the reactions can be done at high volumes andin another embodiment the reactions can be scaled to 100,000 L or more.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Provided herein are methods of making N^(α),N^(α),N^(α)-trialkylhistidine derivative compounds, and methods of their use for makingcompounds such as ergothioneine. Ergothioneine is useful for protectingcells from reactive oxidative species in microbes, plants, and animals,including humans.

DEFINITIONS

When referring to the methods and compounds described herein, thefollowing terms have the following meanings unless indicated otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. In the event that there is a plurality of definitions for aterm herein, those in this section prevail unless stated otherwise.

The term “alkyl,” as used herein, unless otherwise specified, refers toa saturated straight or branched hydrocarbon. In certain embodiments,the alkyl group is a primary, secondary, or tertiary hydrocarbon. Incertain embodiments, the alkyl group includes one to ten carbon atoms,in another embodiment one to four carbon atoms. In certain embodiments,the alkyl group is selected from the group consisting of methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, and t-butyl. The termincludes both substituted and unsubstituted alkyl groups, includinghalogenated alkyl groups. In certain embodiments, the alkyl group is afluorinated alkyl group. In certain embodiments, the alkyl group can besubstituted with at least one (in certain embodiments 1, 2, 3, 4, or 5)group selected from the group consisting of halogen (fluoro, chloro,bromo or iodo), hydroxyl, alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, cycloalkyl, aryl, sulfanyl, alkylsulfanyl,cycloalkylsulfanyl, arylsulfanyl, amino (as defined herein, e.g.,alkylamino, arylamino etc.), alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, sulfonate, phosphonic acid, phosphate, or phosphonate,either unprotected, or protected as necessary, as known to those skilledin the art, for example, as taught in Greene, et al., Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference. In certain embodiments, the alkyl group canbe substituted with 1, 2, 3, 4, or 5 or more substituents describedabove. In certain embodiments, the alkyl group is unsubstituted.

The term “alkoxy” as used herein refers to an —OR group where R is alkylas defined herein. In certain embodiments, alkoxy and alkoxyl groupsinclude, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy,n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,1,2-dimethylbutoxy, and the like.

The term “alkylcarbonyl” as used herein refers to a —C(O)R group where Ris alkyl as defined herein.

The term “alkylsulfanyl” as used herein refers to a —SR group where R isalkyl as defined herein.

“Amino” refers to the group —NRR′ wherein R and R′ are independentlyhydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, orheteroaryl, each of which is as defined herein. In an embodiment,“amino” is —NH₂.

The term “aryl,” as used herein, and unless otherwise specified, refersto a substituent derived from a carbocyclic aromatic ring. In anembodiment, an aryl group is a C₆-C₁₂ aryl group. In an embodiment, anaryl group is phenyl or naphthyl. The term includes both substituted andunsubstituted moieties. An aryl group can be substituted with one ormore moieties (in certain embodiments 1, 2, 3, 4, or 5) independentlyselected from the group consisting of halogen (fluoro, chloro, bromo oriodo), alkyl, haloalkyl, hydroxyl, amino (as defined herein, e.g.alkylamino, arylamino etc.), alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, sulfonate, phosphonic acid, phosphate, and phosphonate,either unprotected, or protected as necessary, as known to those skilledin the art, for example, as taught in Greene, et al., Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991. Incertain embodiments, an aryl group is substituted with 1, 2, 3, 4, 5, ormore moieties described above.

The term “aralkyl” as used herein refers to an alkyl group substitutedwith one or two aryl groups.

The term “arylcarbonyl” as used herein refers to a —C(O)R group where Ris aryl as defined herein.

The term “aryloxy” as used herein refers to a —OR group where R is arylas defined herein.

The term “arylsulfanyl” as used herein refers to an —SR group where R isaryl as defined herein.

The term “cycloalkyl,” as used herein, unless otherwise specified,refers to a saturated monocyclic or polycyclic hydrocarbon. In certainembodiments, cycloalkyl includes fused, bridged, and spiro ring systems.In certain embodiments, the cycloalkyl group includes three to tencarbon atoms, i.e., C₃ to C₁₀ cycloalkyl. In some embodiments, thecycloalkyl has from 3 to 15 (C₃₋₁₅), from 3 to 10 (C₃₋₁₀), or from 3 to7 (C₃₋₇) carbon atoms. In certain embodiments, the cycloalkyl group is,for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclohexylmethyl, cycloheptyl, bicyclo[2.1.1]hexyl,bicyclo[2.2.1]heptyl, decalinyl or adamantyl.

The term “cycloalkylcarbonyl” as used herein refers to a —C(O)R groupwhere R is cycloalkyl as defined herein.

The term “cycloalkylsulfanyl” as used herein refers to a —SR group whereR is cycloalkyl as defined herein.

The term “phosphonic acid” refers to —P(O)(OH)₂.

The term “phosphate” refers to the group —OP(O)(OR)₂ where each R isindependently alkyl or arylalkyl.

The term “phosphonate” refers to the group —P(O)(OR)₂ where each R isindependently alkyl or arylalkyl.

The term “sulfanyl” as used herein refers to a —SH group.

The term “sulfonic acid” refers to the group —S(O)₂OH.

The term “sulfate” refers to the group —OS(O)₂OR where R is alkyl orarylalkyl.

The term “sulfonate” refers to the group —S(O)₂OR where R is alkyl orarylalkyl.

“Salt” refers to any salt of a compound provided herein. Such salts maybe derived from a variety of organic and inorganic counter-ions wellknown in the art. Such salts include, but are not limited to: (1) acidaddition salts formed with organic or inorganic acids such ashydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic,acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic,cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic,succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric,benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic,phthalic, lauric, methanesulfonic, ethanesulfonic,1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic,4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic,camphoric, camphorsulfonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric,gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic,cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2) baseaddition salts formed when an acidic proton present in the parentcompound either (a) is replaced by a metal ion, e.g., an alkali metalion, an alkaline earth ion or an aluminum ion, or alkali metal oralkaline earth metal hydroxides, such as sodium, potassium, calcium,magnesium, aluminum, lithium, zinc, and barium hydroxide, ammonia or (b)coordinates with an organic base, such as aliphatic, alicyclic, oraromatic organic amines, such as ammonia, methylamine, dimethylamine,diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine,ethylenediamine, lysine, arginine, ornithine, choline,N,N′-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine,N-benzylphenethylamine, N-methylglucamine piperazine,tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and thelike.

Salts further include, by way of example only and without limitation,sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium andthe like, and when the compound contains a basic functionality, salts ofnon-toxic organic or inorganic acids, such as hydrohalides, e.g.hydrochloride and hydrobromide, sulfate, phosphate, sulfamate, nitrate,acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate,cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate,malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate,tartarate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate,cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate),ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate,benzenesulfonate (besylate), 4-chlorobenzenesulfonate,2-naphthalenesulfonate, 4-toluenesulfonate, camphorate,camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate,glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluconate, benzoate, glutamate,hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate,muconate and the like.

In certain embodiments, the salt of a compound provided herein retainsits biological properties and is not toxic or otherwise undesirable forpharmaceutical use.

The term “substantially free of” or “substantially in the absence of”with respect to a composition refers to a composition that includes atleast about 85 or 90% by weight, in certain embodiments at least about95%, 98%, 99% or 100% by weight, of a designated enantiomer orstereoisomer of a compound. For example, “substantially free of” or“substantially in the absence of” with respect to a composition canrefer to a composition that includes about 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% by weight of adesignated enantiomer or stereoisomer of a compound. In certainembodiments, in the methods and compounds provided herein, the compoundsare substantially free of other enantiomers or stereoisomers.

Similarly, the term “isolated” with respect to a composition refers to acomposition that includes at least 85, 90%, 95%, 98%, 99% to 100% byweight, of a designated compound, enantiomer, or stereoisomer, theremainder comprising other chemical species, enantiomers, orstereoisomers. For example, “isolated” with respect to a composition canrefer to a composition that includes about 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% by weight of adesignated compound, enantiomer, or stereoisomer, the remaindercomprising other chemical species, enantiomers, or stereoisomers.

Methods

Provided herein are methods of making N^(α),N^(α),N^(α)-trialkylhistidine derivative compounds, and methods of their use for makingcompounds such as ergothioneine. Ergothioneine is useful for protectingcells from reactive oxidative species in microbes, plants, and animals,including humans.

The methods comprise any or all of steps (a), (b), and (c), below. Insome embodiments, the methods comprise step (a). In some embodiments,the methods comprise step (b). In some embodiments, the methods comprisestep (c). In some embodiments, the methods comprise step (a) and step(b). In some embodiments, the methods comprise step (b) and step (c). Insome embodiments, the methods comprise steps (a), (b) and (c).

In one aspect, provided herein are methods comprising step (a). In step(a), a N^(α),N^(α),N^(α)-trialkyl histidine derivative compound isreacted with an acyl chloride or a chloroformate to open its ring:

In Formula 1, each R¹ is (C₁₋₄)alkyl. The R¹ alkyl groups can be thesame or different. In particular embodiments, the alkyl groups are thesame. In particular embodiments, each R¹ is methyl. In Formula 3, eachR¹ is consistent with Formula 1. Preferably, in Formula 1, and Formula3, each R¹ is unsubstituted.

In step (a), the reaction is carried out one or more solvents. Incertain embodiments, the mixture of solvents is a biphasic systemconsisting of water and another solvent not completely miscible withwater. The solvent(s) can be any solvent deemed suitable to those ofskill in the art for carrying out the reaction. In certain embodiments,the solvent(s) is partially miscible with water and does not appreciablyreact with the compound of Formula 2. In certain embodiments, thesolvent(s) is selected from the group consisting of water, ethylacetate, tetrahydrofuran (THF), 2-methyltetrahydrofuran, dioxane, methylethyl ketone, acetone, dimethylformamide, dimethylsulfoxide, diglyme,(bis)-methoxymethyl ether, and (bis)-2-ethoxyethyl ether. In particularembodiments, the solvent is a mixture of THF and water. For, instance,the solvent can be 50% THF and 50% water. In particular embodiments, thesolvent is ethyl acetate. In certain embodiments, the solvent(s) isselected from the group consisting of water, ethyl acetate,tetrahydrofuran (THF), 2-methyltetrahydrofuran, dioxane, methyl ethylketone, acetone, dimethylformamide, diethyl ether, dimethylsulfoxide,diglyme, (bis)-methoxymethyl ether, and (bis)-2-ethoxyethyl ether. Inparticular embodiments, the solvent is a mixture of diethyl ether andwater. In some embodiments, the solvent is 50% diethyl ether and 50%water. In particular embodiments, the solvent is ethyl acetate or THF.

The reaction of step (a) is preferably carried out with a base. Inparticular embodiments, the base is selected from the group consistingof sodium bicarbonate, sodium carbonate, potassium acetate, sodiumhydroxide, sodium acetate, and potassium hydrogen carbonate. Inparticular embodiments, the base is bicarbonate, for example sodiumbicarbonate.

The concentration of the compound according to Formula 1 is about 0.05Mto about 1.2M, in another embodiment about 0.1M in another embodimentabout 0.2M in another embodiment about 0.3M in another embodiment about0.4M in another embodiment about 0.5M in another embodiment about 0.6Min another embodiment about 0.7M in another embodiment about 0.8M, inanother embodiment about 0.9M in another embodiment about 1.0M inanother embodiment about 1.1M, and in another embodiment about 1.2M. Thecompound according to Formula 2 is used in an amount of at least about 2equivalents with respect to the compound of Formula I, and in someembodiments about 2 equivalents, about 2.5 equivalents, about 3equivalents, or about 3.5 equivalents. In particular embodiments, thecompound of Formula 2 is in at least a two-fold molar excess compared tothe compound of Formula 1.

The reaction of step (a) is carried out at any temperature deemedsuitable by those of skill in the art. In particular embodiments, thereaction is conducted at any temperature from about 0° C. to about 30°C., from about 5° C. to about 25° C., from about 10° C. to about 25° C.,from about 18° C. to about 25° C., or from about 20° C. to about 30° C.In particular embodiments, the reaction is at room temperature. Inparticular embodiments, the reaction is conducted at about 20° C., about21° C., about 22° C., about 23° C., about 24° C., or about 25° C.

The reaction of step (a) can be carried out in any volume deemedsuitable by those of skill in the art and depends on the size of thereaction. In particular embodiments, the reaction volume is at leastabout 50 mL, at least about 100 mL, at least about 150 mL, at leastabout 200 mL, at least about 225 mL, at least about 250 mL, at leastabout 500 mL, at least about 1 L, at least about 2 L, at least about 3L, at least about 4 L, or at least about 5 L. In another embodiment, thereaction volume is from at least about 200 mL to at least about 100,000L. In another embodiment, the reaction volume is at least about 1000 L,at least about 5000 L, at least about 10,000 L, at least about 25,000 L,at least about 50,000 L, at least about 75,000 L, or at least about100,000 L.

The reaction of step (a) can proceed for any time deemed suitable forformation of the compound according to Formula 3. In particularembodiments, the reaction proceeds for about 1-48 hours. In particularembodiments, the reaction proceeds for about 1-8 hours, in anotherembodiment about 1-5 hours, in another embodiment about 2-12 hours, andin another embodiment about 5-48 hours. In certain embodiments, thereaction proceeds for about 15-30 hours. In particular embodiments, thereaction proceeds for about 24 hours. In certain embodiments, thereaction proceeds for about 1 hour, about 2 hours, about 3 hours, about4 hours, about 5 hours, or about 6 hours. In particular embodiments, thereaction proceeds for about 1 to about 6 hours, in another embodimentabout 1 to about 4 hours, in another embodiment about 2 to about 4hours, in another embodiment about 2.5 to about 3.5 hours. Reactionprogress can be monitored by standard techniques such as thin layerchromatography or high-performance liquid chromatography.

In certain embodiments, the compound of Formula 3 is isolated from thereaction mixture. The compound can be isolated by any technique deemedsuitable by those of skill. In particular embodiments, side productssuch as an alcohol can be removed by extraction with dichloromethane,ethyl acetate or ether.

In another aspect, provided herein are methods comprising step (b). Oneembodiment of step (b) is step (b)(1). In step (b)(1), a ring-openedcompound of Formula 3 is treated with a catalyst, such as Pd/C or Pd/Csulfided, and hydrogen gas:

In Formula 3, each R¹ is (C₁₋₄)alkyl. The R¹ alkyl groups can be thesame or different. In particular embodiments, the alkyl groups are thesame. In particular embodiments, each R¹ is methyl. In Formula 4, eachR¹ is consistent with Formula 3. Preferably, in Formula 3 and Formula 4,each R¹ is unsubstituted.

The reaction of step (b)(1) is carried out using a catalyst and hydrogengas or alternatively, formic acid, ammonium formate, cyclohexene,cyclohexadiene or the like. The catalyst can be any deemed suitable bythose of skill. In particular embodiments, the catalyst is a Palladiumcatalyst (e.g. Pd/C, Pd/C sulfided, a Lindlar catalyst), a Rhodiumcatalyst (e.g. Wilkinson's catalyst), a Nickel catalyst (e.g. RaneyNickel, Urushibara Nickel), or a Pt catalyst. In some embodiments, thecatalyst is Pd/C, Pd/C sulfided, Pd/CaSO₄, Pd/BaSO₄, Pd/CaCO₃,RhCl(PPh₃)₃, Raney Nickel, Pt/CaSO₄, Pt/BaSO₄, or Pt/CaCO₃; where thePd/CaSO₄, Pd/BaSO₄, Pd/CaCO₃, Pt/CaSO₄, Pt/BaSO₄, and Pt/CaCO₃ catalystsare optionally partially poisoned (in one embodiment optionallypartially poisoned with Pb or 2,6-lutidine). In particular embodiments,the catalyst is Pd/C or Pd/C sulfided. In some embodiments, the amountof catalyst employed to carry out the reaction is about 0.2 mol % toabout 20 mol %, about 0.25 mol % to about 10 mol %, about 0.5 mol % toabout 7.5 mol %, about 1 mol %, about 2 mol %, about 3 mol %, about 4mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about9 mol %, or about 10 mol %. In certain embodiments, the amount ofcatalyst is about 0.2 mol % to about 20 mol % Pd/C, about 0.25 mol % toabout 10 mol % Pd/C, about 0.5 mol % to about 7.5 mol % Pd/C, about 1mol % Pd/C, about 2 mol % Pd/C, about 3 mol % Pd/C, about 4 mol % Pd/C,about 5 mol % Pd/C, about 6 mol % Pd/C, about 7 mol % Pd/C, about 8 mol% Pd/C, about 9 mol % Pd/C, or about 10 mol % Pd/C.

In step (b)(1), the reaction is carried out in one or more solvent(s).The solvent(s) can be any solvent deemed suitable to those of skill inthe art for carrying out the reaction. In certain embodiments, thesolvent(s) is selected from the group consisting of dilute hydrochloricacid (in one embodiment about 1%), water, methanol, ethanol, andcombinations thereof. In particular embodiments, the solvent is water.

In step (b)(1), the concentration of the compound according to Formula 3is preferably from about 1 g/100 mL to about 10 g/100 mL, in anotherembodiment about 3 g/100 mL to about 6 g/100 mL, and in anotherembodiment about 4 g/100 mL to about 5 g/100 mL. In another embodiment,the concentration is about 1 g/100 mL, about 2 g/100 mL, about 3 g/100mL, about 4 g/100 mL, about 5 g/100 mL, about 6 g/100 mL, about 7 g/100mL, about 8 g/100 mL, about 9 g/100 mL, or about 10 g/100 mL. The amountof catalyst is preferably from about 0.25 mol % Pd/C to about 7.5 mol %Pd/C.

The reaction of step (b)(1) is carried out at any temperature deemedsuitable by those of skill in the art. In particular embodiments, thereaction is conducted at any temperature from about 20° C. to about 30°C., from about 20° C. to about 28° C., or from about 20° C. to about 25°C. In particular embodiments, the reaction is at room temperature. Inparticular embodiments, the reaction is conducted at about 20° C., about21° C., about 22° C., about 23° C., about 24° C., or about 25° C.

The reaction of step (b)(1) can be carried out in any volume deemedsuitable by those of skill in the art. In particular embodiments, thereaction volume is at least about 500 mL, at least about 1 L, at leastabout 2 L, at least about 3 L, at least about 4 L, or at least about 5L. In another embodiment, the reaction volume is from at least about 500mL to at least about 100,000 L. In another embodiment, the reactionvolume is at least about 1000 L, at least about 5000 L, at least about10,000 L, at least about 25,000 L, at least about 50,000 L, at leastabout 75,000 L, or at least about 100,000 L.

The reaction of step (b)(1) can proceed for any time deemed suitable forformation of the compound according to Formula 4. In particularembodiments, the reaction proceeds for about 1 hour, about 2 hours,about 3 hours, about 4 hours, about 5 hours, about 6 hours, or about 7hours. In particular embodiments, the reaction proceeds for about 1 toabout 6 hours, in another embodiment about 3 to about 6 hours. Reactionprogress can be monitored by standard techniques such as thin layerchromatography or high-performance liquid chromatography.

Another embodiment of step (b) is step (b)(2). In Step (b)(2), aring-opened compound of Formula 3 is transformed to a to compound ofFormula 4 by hydrolysis and rearrangement using an acid. The acid can beany acid deemed useful by the practitioner of skill. Useful acidsinclude hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid,boric acid, hydrofluoric acid, hydrobromic acid, and trifluoroaceticacid.

In step (b)(2), the concentration of the compound according to Formula 3can be any concentration deemed useful by the practitioner of skill. Incertain embodiments, the concentration is at least about 1 g/10 mL. Incertain embodiments, the concentration is from about 1 g/10 mL to about1 g/mL, or from about 1 g/10 mL to about 5 g/10 mL, or from about 1 g/10mL to about 3 g/10 mL. In particular embodiments, the concentration isabout 1 g/10 mL, 2 g/10 mL, 3 g/10 mL, 4 g/10 mL, or 5 g/10 mL, 6 g/10mL, 7, g/10 mL, 8 g/10 mL, 9 g/10 mL or 1 g/mL.

In step (b)(2), the concentration of acid is any amount deemedsufficient for the hydrolysis and rearrangement to proceed. In certainembodiments, the acid concentration is from about 1% to about 38% (w/v).In certain embodiments, the acid concentration is from about 5% to about38% (w/v), or from about 10% to about 38% (w/v), from about 15% to about38% (w/v), or from about 20% to about 38% (w/v). In certain embodiments,the acid concentration is from about 20% to about 38% (w/v). The acidcan be in any solvent deemed suitable, for instance, water. Inparticular embodiments, the reaction proceeds in aqueous acid. Incertain embodiments, the reaction proceeds in aqueous acid with animmiscible solvent. The immiscible solvent can advantageously extractthe benzyl chloride side product. Useful immiscible solvents includeethyl acetate, ether, dichloromethane, and the like. The ratio ofimmiscible solvent to aqueous solution can be any ratio deemed suitable.In some embodiments, the ratio of immiscible solvent to aqueous solutioncan be from about 1:10 to about 10:1, or from about 1:5 to about 5:1, orfrom about 1:3 to about 3:1, or from about 1:2 to about 2:1, or about1:1. In some embodiments, the ratio is about 3:10 immiscible solvent toaqueous solution. The reaction volume can be any volume deemed useful bythe practitioner of skill. In certain embodiments, the reaction volumecan range from 25 mL to 40 L or greater. In certain embodiments, thereaction volume is at least about 25 mL, 50 mL, 100 mL, 250 mL, 500 mL,1 L, 2 L, 5 L, 10 L, 20 L, 25 L, 30 L, 35 L, or 40 L.

In step (b)(2), the reaction can be carried out at any temperaturedeemed suitable by those of skill in the art. In certain embodiments,the reaction is carried out at a temperature of about 0° C. to about 40°C. In certain embodiments, the reaction is carried out at a temperatureof about 5° C. to about 35° C. In certain embodiments, the reaction iscarried out at a temperature of about 10° C. to about 30° C. In anotherembodiment, the reaction is carried out at a temperature of about 15° C.to about 25° C.

In step (b)(2), the reaction can proceed for any time deemed suitablefor formation of the compound according to Formula 4. In certainembodiments, the time for reaction is from 24 hours to about 48 hours.In certain embodiments, the time for reaction is from about 1 hour toabout 24 hours. In certain embodiments, the time for reaction is from 12hours to about 24 hours. In certain embodiments, the time for reactionis from 12 hours to about 18 hours. In certain embodiments, the time forreaction is from 3 hours to about 15 hours. In certain embodiments, thetime for reaction is from 8 hours to about 12 hours.

In certain embodiments, the compound of Formula 4 is isolated from thereaction mixture. The compound can be isolated by any technique deemedsuitable by those of skill. In particular embodiments, the compound ofFormula 4 is isolated by filtration and evaporation. In particularembodiments, the intermediate is used in the next step without furtherisolation or concentration. In further embodiments, the compound ofFormula 4 is isolated by evaporation and optional filtration, followedby dissolving in a solvent for step (c) below. As described below, theuseful solvents include hydrochloric acid in water or ethanol, or both.

In another aspect, provided herein are methods comprising step (c). Instep (c), a compound of Formula 4 is reacted with a thiocyanate to yieldthe product according to Formula 5:

In Formula 4, each R¹ is (C₁₋₄)alkyl. The R¹ alkyl groups can be thesame or different. In particular embodiments, the alkyl groups are thesame. In particular groups, each R¹ is methyl. In Formula 5, each R¹ isconsistent with Formula 4. Preferably, in Formula 4 and Formula 5, eachR¹ is unsubstituted.

The reaction of step (c) is carried out with a thiocyanate. Thethiocyanate can be any thiocyanate deemed suitable by those of skill. Inparticular embodiments, the agent is selected from the group consistingof potassium thiocyanate (KCNS), lithium thiocyanate (LiCNS), ammoniumthiocyanate (NH₄CNS), and sodium thiocyanate (NaCNS). In particularembodiments, the thiocyanate is KCNS. In particular embodiments, thethiocyanate is ammonium thiocyanate (NH₄CNS).

In step (c), the reaction is carried out in one or more solvent(s). Thesolvent(s) can be any solvent deemed suitable to those of skill in theart for carrying out the reaction. In certain embodiments, thesolvent(s) is dilute hydrochloric acid (in one embodiment about 1%(w/v)) and water. In certain embodiments, the solvent(s) is hydrochloricacid (in certain embodiments from about 1% (w/v) to about 38% (w/v)) andwater or ethanol, or both. In certain embodiments, the solvent(s) iswater, 1-38% (w/v) hydrochloric acid, methanol, ethanol, isopropanol, ora combination thereof. In particular embodiments, the solvent is water.

The concentration of the compound according to Formula 4 is from about0.5 g/25 mL to about 5 g/25 mL, in another embodiment from about 1 g/25mL to about 4 g/25 mL. The amount of thiocyanate can be any amountdeemed suitable by the practitioner of skill. In certain embodiments,the amount of thiocyanate is from about 1 to about 3 equivalents,relative to the amount of the compound of Formula 4. In certainembodiments, the amount of thiocyanate is from about 1 g/25 mL to about5 g/25 mL. In particular embodiments, the amount of thiocyanate is about1 g/5 mL. In some embodiments, the amount of thiocyanate is about 0.5g/25 mL, 1 g/25 mL, 2 g/25 mL, 3 g/25 mL, or 4 g/25 mL.

The reaction of step (c) is carried out at any temperature deemedsuitable by those of skill in the art. In particular embodiments, thereaction is conducted at any temperature from about room temperature toabout 95° C. or to about 100° C. In particular embodiments, the reactionis conducted at any temperature from about 40° C. to about 95° C. Inparticular embodiments, the reaction is conducted at any temperaturefrom about 50° C. to about 95° C. In particular embodiments, thereaction is conducted at any temperature from about 60° C. to about 95°C. In particular embodiments, the reaction is conducted at anytemperature from about 70° C. to about 95° C. In particular embodiments,the reaction is conducted at any temperature from about 80° C. to about95° C. In particular embodiments, the reaction is conducted at anytemperature from about 85° C. to about 95° C. In particular embodiments,the reaction is at any temperature from about 85° C. to about 90° C.

The reaction of step (c) can be carried out in any volume deemedsuitable by those of skill in the art. In particular embodiments, thereaction volume is at least about 10 mL, at least 20 mL, at least 25 mL,at least 50 mL, at least 60 mL, at least 70 mL, at least 80 ml, at least90 mL, or at least 100 mL. In another embodiment, the reaction volume isat least about 10 mL to at least about 100,000 L. In another embodiment,the reaction volume is at least about 1000 L, at least about 5000 L, atleast about 10,000 L, at least about 25,000 L, at least about 50,000 L,at least about 75,000 L, or at least about 100,000 L.

The reaction of step (c) can proceed for any time deemed suitable forformation of the compound according to Formula 5. In particularembodiments, the reaction proceeds for about 0.5 hours, about 1 hour,about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about3.5 hours, about 4 hours, about 5 hours, about 6 hour 7 hours, about 8hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours,about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, or about 24 hours. In particularembodiments, the reaction proceeds for about 0.5 to about 2 hours and inanother embodiment from about 0.5 to about 1.5 hours. In particularembodiments, the reaction is conducted at low temperature (in anotherembodiment, at any temperature from about 20° C. to about 100° C., inanother embodiment, at any temperature from about 20° C. to about 60°C., in another embodiment at about 20° C., in another embodiment atabout room temperature, in another embodiment at about 30° C., inanother embodiment at about 35° C., in another embodiment at about 40°C., in another embodiment at about 45° C., in another embodiment atabout 50° C., in another embodiment at about 55° C., in anotherembodiment at about 60° C., and in another embodiment at 100° C.) and isallowed to proceed overnight or about 18-24 hours. Reaction progress canbe monitored by standard techniques such as thin layer chromatography orhigh-performance liquid chromatography.

In certain embodiments, the compound of Formula 5 is isolated from thereaction mixture. The compound can be isolated by any technique deemedsuitable by those of skill. In particular embodiments, the compound ofFormula 5 is isolated by filtration and evaporation and crystallization.

The present disclosure encompasses methods that comprise any of steps(a), (b), and (c). In particular embodiments, provided herein aremethods of making compounds according to Formula 5 by following steps(a), (b), and (c) above. In further embodiments, provided herein aremethods of making compounds according to Formula 5, wherein each R¹ ismethyl, by following steps (a), (b), and (c) above. In such embodiments,the compound according to Formula 1 is hercynine and the compoundaccording to Formula 5 is ergothioneine.

The compounds of Formulas 1, 3, 4, and 5 each include a chiral center.The present specification encompasses methods using compounds with anystereochemistry at the chiral centers. In particular embodiments, thecompounds are racemic. In particular embodiments, the compounds haveD-stereochemistry. In preferred embodiments, the compounds haveL-stereochemistry.

In particular embodiments, the methods of step (a) are carried out witha compound according to Formula 1a:

or a salt or tautomer thereof.

In particular embodiments, the methods of step (a) yield a compoundaccording to Formula 3a:

or a salt thereof.

In particular embodiments, the methods of step (b) are carried out witha compound according to Formula 3a:

or a salt thereof.

In particular embodiments, the methods of step (b) yield a compoundaccording to Formula 4a:

or a salt thereof.

In particular embodiments, the methods of step (c) are carried out witha compound according to Formula 4a:

or a salt thereof.

In particular embodiments, the methods of step (c) yield a compoundaccording to Formula 5a:

or a salt or tautomer thereof.

In particular embodiments, the methods of step (a) are carried out witha compound according to Formula 1b:

or a salt or tautomer thereof.

In particular embodiments, the methods of step (a) yield a compoundaccording to Formula 3b:

or a salt thereof.

In particular embodiments, the methods of step (b) are carried out witha compound according to Formula 3b:

or a salt thereof.

In particular embodiments, the methods of step (b) yield a compoundaccording to Formula 4b:

or a salt thereof.

In particular embodiments, the methods of step (c) are carried out witha compound according to Formula 4b:

or a salt thereof.

In particular embodiments, the methods of step (c) yield a compoundaccording to Formula 5b:

or a salt or tautomer thereof.

Compounds

Provided herein are compounds useful in the preparation of histidinederivatives such as ergothioneine. In certain embodiments, providedherein are compounds according to Formula 3:

or salts thereof; wherein each R¹ is (C₁₋₄)alkyl. In particularembodiments, the (C₁₋₄)alkyl are unsubstituted. In particularembodiments, each R¹ is methyl.

Provided herein are compounds useful in the preparation of histidinederivatives such as ergothioneine. In certain embodiments, providedherein are compounds according to Formula 3a:

or salts thereof wherein each R¹ is (C₁₋₄)alkyl. In particularembodiments, the (C₁₋₄)alkyl are unsubstituted. In particularembodiments, each R¹ is methyl.

Provided herein are compounds useful in the preparation of histidinederivatives such as ergothioneine. In certain embodiments, providedherein are compounds according to Formula 3b:

or salts thereof wherein each R¹ is (C₁₋₄)alkyl. In particularembodiments, the (C₁₋₄)alkyl are unsubstituted. In particularembodiments, each R¹ is methyl.

In certain embodiments, provided herein are compounds according toFormula 4:

or salts thereof wherein each R¹ is (C₁₋₄)alkyl. In particularembodiments, the (C₁₋₄)alkyl are unsubstituted. In particularembodiments, each R¹ is methyl.

In certain embodiments, provided herein are compounds according toFormula 4a:

or salts thereof; wherein each R¹ is (C₁₋₄)alkyl. In particularembodiments, the (C₁₋₄)alkyl are unsubstituted. In particularembodiments, each R¹ is methyl.

In certain embodiments, provided herein are compounds according toFormula 4b:

or salts thereof; wherein each R¹ is (C₁₋₄)alkyl. In particularembodiments, the (C₁₋₄)alkyl are unsubstituted. In particularembodiments, each R¹ is methyl.

In certain embodiments, provided herein are compounds according toFormula 5:

or salts thereof; wherein each R¹ is (C₁₋₄)alkyl. In particularembodiments, the (C₁₋₄)alkyl are unsubstituted. In particularembodiments, each R¹ is methyl.

In certain embodiments, provided herein are compounds according toFormula 5a:

or salts thereof; wherein each R¹ is (C₁₋₄)alkyl. In particularembodiments, the (C₁₋₄)alkyl are unsubstituted. In particularembodiments, each R¹ is methyl.

In certain embodiments, provided herein are compounds according toFormula 5b:

or salts thereof; wherein each R¹ is (C₁₋₄)alkyl. In particularembodiments, the (C₁₋₄)alkyl are unsubstituted. In particularembodiments, each R¹ is methyl.

Particular compounds include the following:

and salts thereof.

EXAMPLES

As used herein, the symbols and conventions used in these processes,schemes and examples, regardless of whether a particular abbreviation isspecifically defined, are consistent with those used in the contemporaryscientific literature, for example, the Journal of the American ChemicalSociety or the Journal of Biological Chemistry. Specifically, butwithout limitation, the following abbreviations may be used in theexamples and throughout the specification: g (grams); mg (milligrams);mL (milliliters); μL (microliters); M (or M, molar); mM (millimolar); μM(micromolar); Hz (Hertz); MHz (megahertz); mol (moles); mmol(millimoles); hr or hrs (hours); min (minutes); MS (mass spectrometry);ESI (electrospray ionization); TLC (thin layer chromatography); HPLC(high pressure liquid chromatography); THF (tetrahydrofuran); CDCl₃(deuterated chloroform); AcOH (acetic acid); DCM (dichloromethane); DMSO(dimethylsulfoxide); DMSO-d₆ (deuterated dimethylsulfoxide); EtOAc(ethyl acetate); MeOH (methanol); RVE (rotary evaporator); and BOC(t-butyloxycarbonyl).

For all of the following examples, standard work-up and purificationmethods known to those skilled in the art can be utilized. Unlessotherwise indicated, all temperatures are expressed in ° C. (degreesCentigrade). All reactions are conducted at room temperature unlessotherwise noted. Synthetic methodologies illustrated herein are intendedto exemplify the applicable chemistry through the use of specificexamples and are not indicative of the scope of the disclosure.

Example 1 Preparation of L-Ergothioneine (E)

Compound C

To a 1 liter 3-neck flask with motor-stirrer, thermometer and 50 mLaddition funnel was added 16 g (80 mmol) of hercynine (A), 300 mL water,40 g (475 mmol) sodium bicarbonate and 300 mL THF. Under strong stirringwas slowly added at room temperature over 15-20 minutes, drop wise, 30mL (35.85 g, 210 mmol) CBZ-chloride (B).

After another 2 hours, a sample was analyzed by HPLC for the formationof product (C) in the form of C, C1 or C2, and little or no startingmaterial (hercynine A or CBZ-chloride (B), or both). If startingmaterials remained, the reaction continued. When little or no startingmaterial was present as analyzed by HPLC, small portions of dilute HClwere added to the mixture while stirring to achieve pH 4 to 5, and thecontents of the reaction flask were transferred to a rotary evaporatorflask.

Under vacuum and bath temperature 30° C., THF was removed and the restin the flask (water) was transferred to a 1 liter separatory funnel. Theside product—benzyl alcohol—was removed by extraction with ethyl acetateor ether.

After evaporation of the aqueous layer (RVE, 1 liter flask, 30° C. bath,vacuum) the oily residue was dissolved in methanol. After the insolublematerial was removed by filtration. deformylation was carried out byheating for 2 hours at 64° C. (60-65° C.) in 500 mL of methanol on RVE.Reaction progress was checked by HPLC. When ready, the methanol wasremoved at 30-33° C. under vacuum to a constant weight. The residue wasmixed with EtOH (200 mL) and evaporated to constant weight. The residuewas stirred with 500 mL of DCM for 30 minutes at 30° C. and thenfiltered. The filtrate was evaporated and the product was recrystallizedfrom IPA/ether

After drying in open air compound C was produced as white to yellowcrystals. Yield is 30 g (82%).

Compound D

5 g of compound C from step 1 was dissolved in 100 mL of 1% HCl. To thiswas added 0.1 g of 5% Pd/C Sulfided. To this solution/mixture undermagnetic stirring in a 500 mL 3-neck flask was bubbled hydrogen gasuntil the evolution of CO₂ ceased, approximately 3 hours.

When reaction progress was complete by HPLC, the palladium was filteredoff and the filtrate was used without purification in the next step.

The yield by weight is approximately 100%.

L-Ergothioneine (E)

The solution of compound D (approximately 11 mmol) from step 2 waswarmed to 85-90° C. and 5 g of KCNS in 10 mL water was added. Thissolution was stirred in a water bath at 85-90° C. for 1-2 hours.

When reaction progress was complete by HPLC, this solution was cooled toapproximately 30° C. Approximately 15 mL of concentrated HCl in 15 mL ofwater was added, and this acidic solution was evaporated (bath 30° C.,vacuum). The residue was dissolved in 20 mL ethanol (200 proof) and wasagain evaporated. 30 mL ethanol (200 proof) was added. For a few minutesthis mixture was stirred on bath at 30° C., and then cooled for 20 minon ice. The salts were filtered off and washed with 20 mL of ethanol(200 proof) and the filtrates were basified with a dilute solution ofLiOH to pH 3.6-4 by pH meter.

After total evaporation under vacuum in order to remove water, theresidue was stirred on RVE overnight with 30 mL ethanol. Then the solidwas filtered, washed with 2×5 mL ethanol and dried. Approximately 0.8 gof solid crystalline L-ergothioneine was obtained. Analysis by HPLCshowed approximately 85% pure product. Optical rotation wasapproximately 80-90°, and the color was light brown. From motherliquors, an additional 0.3 g was obtained, for a total 1.1 g (43%) ofL-ergothioneine.

The yield of L-ergothioneine (E) from hercynine (A) is approximately40-50%.

Example 2 Compound C

Example 2 proceeds according to Example 1 with the following exceptions.To obtain Compound C directly, with little or no C1 or C2, the reactionof A and B proceeds with stirring for 24-48 hours. The deformylation ofC1 or C2 to C can proceed to completion or near completion. When byanalysis the process is ready it is then acidified by dilute HCl to pH4-5, transferred to a separatory funnel and the side product of benzylalcohol is removed by extraction with ethyl acetate or diethyl ether.After evaporation of the water layer under vacuum (bath a 40° C.) theremaining material is dissolved in 300 mL dichloromethane, insolublematerial is then removed by filtration, and the filtrate is evaporatedunder vacuum (bath at 35° C.). The product is recrystallized fromIPA-diethyl ether, and after drying in open air compound C is isolatedas off-white crystals. Yield is 32 g (82%).

Example 3 Compound D, by Acid Hydrolysis and Rearrangement

Example 3 proceeds according to Example 1 with the following exceptions.5 g of compound C from step 1 is dissolved in 150 mL of concentrated HCland this solution is stirred at room temperature for 12-24 hours. Whenby analysis the reaction is complete, the mixture is evaporated todryness under vacuum (less than 1 torr) at a bath temperature of lessthan 30° C. The side product of benzyl chloride is distilled off withwater, or can be extracted by suitable solvent, for example bydichloromethane. Yield is 95%.

The product after evaporation is worked up promptly in the followingstep for Compound E according to Example 1. Approximately 1.3 g of solidcrystalline L-ergothioneine was obtained. Analysis by HPLC showedapproximately 85% pure product. Optical rotation was approximately80-90°, and the color was light brown. From mother liquors, anadditional 0.3 g was obtained, for a total 1.6 g of L-ergothioneine.After recrystallization from water-ethanol 1.3 g (51%) of whitecrystalline Ergothioneine (E) was obtained; specific opticalrotation+126° (c=1, 1N HCl), and confirmed by HPLC-Mass Spectrum and NMRanalysis.

Example 4 Compound D, by Acid Hydrolysis and Rearrangement

Example 4 proceeds according to Example 1 with the following exceptions.50 g of compound C from step 1 is dissolved in 150 mL ofdichloromethane. 500 ml of concentrated HCl is added, and this solutionis stirred at room temperature for 12-24 hours. When by analysis thereaction is complete, the mixture is poured into a separatory funnel,the layers separated, and the aqueous phase extracted once more with 100ml of dichloromethane. After separation of layers, the product (compoundD) in aqueous acid is used as is in the final step.

To 100 ml of solution of compound D, 300 ml of distilled water is addedfollowed by 4 g of NH4CNS dissolved in 10 ml of distilled water. Theresulting solution is heated for 6 hours at 85-90 C, cooled to roomtemperature and then worked up as in example 1. Approximately 2.4 g ofcrude Ergothioneine (E) was obtained.

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication, patent, or patent application were specifically andindividually indicated to be incorporated by reference. While theclaimed subject matter has been described in terms of variousembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the claimed subject matter is limited solely by the scope ofthe following claims, including equivalents thereof.

1. A method of producing a compound of Formula 3:

comprising: (a) reacting a compound of Formula 1 with a compound ofFormula 2 to form the compound of Formula 3:

wherein each R¹ is (C₁₋₄)alkyl. 2-35. (canceled)
 36. The method of claim1, further comprising: (b) treating the compound of Formula 3 withhydrogen gas, formic acid, or ammonium formate in the presence of acatalyst; or with an acid to form a compound of Formula 4:

and (c) reacting the compound of Formula 4 with a thiocyanate to form acompound of Formula 5:


37. The method of claim 1, wherein: the compound according to Formula 1is:

and the compound according to Formula 3 is:


38. The method of claim 1, wherein each (C₁₋₄)alkyl is unsubstituted.39. The method of claim 1, wherein each R¹ is methyl.
 40. The method ofclaim 1, wherein step (a) is conducted in one or more solvent(s)selected from the group consisting of water, ethyl acetate,tetrahydrofuran (THF), 2-methyltetrahydrofuran, dioxane, methyl ethylketone, acetone, dimethylformamide, dimethylsulfoxide, diglyme,(bis)-methoxymethyl ether, (bis)-2-ethoxyethyl ether, and diethyl ether.41. The method of claim 1, wherein step (a) is conducted with a baseselected from the group consisting of sodium bicarbonate, sodiumcarbonate, potassium acetate, sodium hydroxide, sodium acetate, andpotassium hydrogen carbonate.
 42. The method of claim 1, wherein step(a) is conducted at a temperature ranging from about 0° C. to about 30°C.
 43. The method of claim 1, wherein step (a) is conducted for about 1to 48 hours.
 44. The method of claim 1, wherein compound 3 is isolatedafter step (a).
 45. The method of claim 36, wherein: the compoundaccording to Formula 4 is:

and the compound according to Formula 5 is:


46. The method of claim 36, wherein step (b) is conducted in a solventselected from the group consisting of water, 1% (w/v) HCl, methanol,ethanol, and 10% to 38% (w/v) HCl.
 47. The method of claim 46, whereinstep (b) is conducted in the presence of a catalyst, and the catalyst isselected from the group consisting of Pd/C, Pd/C sulfided, Pd/CaSO₄,Pd/CaCO₃, Pd/CaCO₃/Pb and Raney Nickel.
 48. The method of claim 47,wherein step (b) is conducted in the presence of a reagent selected fromthe group consisting of hydrogen gas, formic acid, and ammonium formate.49. The method of claim 36, wherein step (b) is conducted in 1% to 38%(w/v) HCl, at a temperature ranging from about 0° C. to about 40° C. andfor a time ranging from about 1 hour to 24 hours.
 50. The method ofclaim 49, wherein step (b) is conducted at a temperature ranging fromabout 20° C. to about 30° C.
 51. The method of claim 49, wherein step(b) is conducted for a time ranging from about 1 hour to about 6 hours.52. The method of claim 36, wherein step (c) is conducted in a solventselected from the group consisting of water, 1% to 38% (w/v) HCl,methanol, ethanol, isopropanol, and combinations thereof.
 53. The methodof claim 36, wherein the thiocyanate of step (c) is selected from thegroup consisting of KCNS, LiCNS, NH₄CNS, and NaCNS.
 54. The method ofclaim 36, wherein step (c) is conducted at a temperature ranging fromabout room temperature to about 95° C. and for a time ranging from about1 hour to about 24 hours.
 55. The method of claim 54, wherein step (c)is conducted at a temperature ranging from about 20° C. to about 60° C.56. The method of claim 54, wherein step (c) is conducted for a timeranging from about 1 hour to about 4 hours.
 57. The method of claim 36,wherein the compound according to Formula 5 is isolated after step (c).58. A method of producing a compound of Formula 5:

comprising: (c) reacting a compound of Formula 4 with a thiocyanate toform the compound of Formula 5:

wherein each R¹ is (C₁₋₄)alkyl.
 59. The method of claim 58, wherein step(c) is conducted in a solvent selected from the group consisting ofwater, 1% to 38% (w/v) HCl, methanol, ethanol, isopropanol, andcombinations thereof.
 60. The method of claim 58, wherein thethiocyanate of step (c) is selected from the group consisting of KCNS,LiCNS, NH₄CNS, and NaCNS.
 61. The method of claim 58, wherein step (c)is conducted at a temperature ranging from about room temperature toabout 95° C. and for a time ranging from about 1 to about 24 hours. 62.The method of claim 61, wherein step (c) is conducted at a temperatureranging from about 20° C. to about 60° C.
 63. The method of claim 61,wherein step (c) is conducted for a time ranging from about 1 hour toabout 4 hours.
 64. The method of claim 58, wherein the compoundaccording to Formula 5 is isolated after step (c).